Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer.

Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.

Gene expression analysis in prostate cancer: the importance of the endogenous control.

BACKGROUND: Aberrant gene expression is a hallmark of cancer. Quantitative reverse-transcription PCR (qRT-PCR) is the gold-standard for quantifying gene expression, and commonly employs a house-keeping gene (HKG) as an endogenous control to normalize results; the choice of which is critical for accurate data interpretation. Many factors, including sample type, pathological state, and oxygen levels influence gene expression including putative HKGs. The aim of this study was to determine the suitability of commonly used HKGs for qRT-PCR in prostate cancer. METHODS: Prostate cancer (LNCaP, 22Rv1, PC3, and DU145) and normal (PWR1E and RWPE1) cell lines were cultured in air and hypoxia. The performance of 16 HKGs was assessed using Normfinder and coefficient of variation. In silico promoter analysis was performed to identify putative hypoxia response elements (HREs). The impact of the endogenous control on expression levels of HIF1A and GSTP1 was investigated by qRT-PCR in cell lines and tissue specimens respectively. RESULTS: Hypoxia altered expression of several HKGs: IPO8, B2M, and PGK1. The most stably expressed HKGs were ACTB, PPIA, and UBC. Both UBC and ACTB showed constitutive expression of HIF1A in air and hypoxia, while PGK1 falsely implied a sixfold hypoxia-induced down-regulation. In prostate tumors, UBC and PGK1 both revealed down-regulation of GSTP1 relative to matched benign, whereas ACTB showed variability. CONCLUSIONS: This study demonstrates that no universal endogenous control exists for gene expression studies, even within one disease type. It highlights the importance of validating expression of intended HKGs between different sample types and environmental exposures.

IGFBP7 promoter methylation and gene expression analysis in prostate cancer.

PURPOSE: IGFBP7 belongs to a family of insulin-like growth factor-1 regulatory binding proteins. IGFBP7 hypermethylation is associated with its down-regulation in various carcinomas. In prostate cancer IGFBP7 down-regulation has been widely reported but to our knowledge the mechanisms behind this event are unknown. We performed a denaturing high performance liquid chromatography screening and validation strategy to profile the methylation status of IGFBP7 in prostate cancer. MATERIALS AND METHODS: We combined denaturing high performance liquid chromatography and bisulfite sequencing to examine IGFBP7 methylation in a panel of prostate cancer cell lines. Quantitative methylation specific polymerase chain reaction was used to determine methylation levels in prostate tissue specimens of primary prostate cancer, histologically benign prostate adjacent to tumor, high grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. IGFBP7 gene expression was measured by quantitative methylation specific polymerase chain reaction in cell lines and tissue specimens. RESULTS: IGFBP7 was methylated in the 4 prostate cancer cell lines DU145, LNCaP, PC-3 and 22RV1. Quantitative methylation specific polymerase chain reaction analysis revealed that promoter methylation was associated with decreased IGFBP7 expression. Quantitative methylation specific polymerase chain reaction showed that IGFBP7 methylation was more frequently detected in prostate cancer (60% (31/52)) and high grade prostatic intraepithelial neoplasia (40% (6/15)) samples compared to histologically benign prostate adjacent to tumor (10%) and benign prostatic hyperplasia (0%) samples. CONCLUSIONS: To our knowledge this is the first report of aberrant IGFBP7 promoter hypermethylation and concurrent IGFBP7 gene silencing in prostate cancer cell lines. Results demonstrate that CpG methylation of IGFBP7 may represent a novel biomarker of prostate cancer and pre-invasive neoplasms. Thus, future examination of IGFBP7 methylation and expression in a larger patient cohort, including bodily fluids, is justified to further evaluate its role in a diagnostic and prognostic setting.

Androgen hypersensitivity in prostate cancer: molecular perspectives on androgen deprivation therapy strategies.

Androgen deprivation therapy is initially successful in treating advanced prostate cancer. However, after a period of time tumors inevitably recur. Improved understanding of the various biochemical causes of resistance to hormonal therapy is of crucial importance for developing more effective therapeutic strategies in this cohort of patients. This review discusses the preclinical evidence for androgen hypersensitivity (AH), as a mechanism by which tumors become hormone-refractory (HR). We propose that the growth of some such tumors may be not only stimulated by, but also dependent on low hormone levels, and furthermore, that normal hormone concentrations can have an inhibitory effect on growth. The incidence and importance of AH merits further investigation both in preclinical studies and during clinical trials of intermittent androgen withdrawal or testosterone replacement. We suggest that a subset of HR prostate cancer patients who have androgen-hypersensitive tumors could be particularly amenable to these treatments. Finally, potential approaches for developing biomarkers to identify such patients are explored.

In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer.

BACKGROUND: Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC). METHODS: Based on an in silico selection process, 13 genes were screened for methylation in CaP cell lines using DHPLC. Quantitative methylation specific PCR was employed to determine methylation levels in prostate tissue specimens (n = 135), representing tumor, histologically benign prostate, high-grade prostatic intraepithelial neoplasia and benign prostatic hyperplasia. Gene expression was measured by QRT-PCR in cell lines and tissue specimens. RESULTS: The promoters of BIK, BNIP3, cFLIP, TMS1, DCR1, DCR2, and CDKN2A appeared fully or partially methylated in a number of malignant cell lines. This is the first report of aberrant methylation of BIK, BNIP3, and cFLIP in CaP. Quantitative methylation analysis in prostate tissues identified 5 genes (BNIP3, CDKN2A, DCR1, DCR2 and TMS1) which were frequently methylated in tumors but were unmethylated in 100% of benign tissues. Furthermore, 69% of tumors were methylated in at least one of the five-gene panel. In the case of all genes, except BNIP3, promoter hypermethylation was associated with concurrent downregulation of gene expression. CONCLUSION: Future examination of this "CaP apoptotic methylation signature" in a larger cohort of patients is justified to further evaluate its value as a diagnostic and prognostic marker.

Differential pathologic variables and outcomes across the spectrum of adenocarcinoma of the esophagogastric junction.

BACKGROUND: Adenocarcinoma of the esophagogastric junction (AEG) as described by Siewert et al. is classified as one entity in the latest (7th Edition) American Joint Cancer Committee/International Union Against Cancer (AJCC/UICC) manual, compared with the previous mix of esophageal and gastric staging systems. The origin of AEG tumors, esophageal or gastric, and their biology remain controversial, particularly for AEG type II (cardia) tumors. METHODS: We adapted a large prospective database (n = 520: 180 type I, 182 type II, 158 type III) to compare AEG tumors under the new TNM system Pathological variables associated with prognosis were compared (pT, pN, stage, differentiation, R status, lymphovascular invasion, perineural involvement, number of positive nodes, percent of positive nodes, and tumor length), as well as overall survival. RESULTS: Compared with AEG type I tumors, type II and type III tumors had significantly (p < 0.05) more advanced pN stages, greater number and percentage of positive nodes, poorer differentiation, more radial margin involvement, and more perineural invasion. In AEG type I, 14/180 patients (8%) had >6 involved nodes (pN3), compared with 16 and 30% of patients classified type II and III, respectively. Median survival was significantly (p = 0.03) improved for type I patients (38 months) compared with those with tumors classified as type II (28 months) and type III (24 months). In multivariate analysis node positivity and pN staging but not AEG site had an impact on survival. CONCLUSIONS: In this series AEG type I is associated with more favorable pathologic features and improved outcomes compared with AEG type II and III. This may reflect earlier diagnosis, but an alternative possibility, that type I may be a unique paradigm with more favorable biology, requires further study.

Gene expression analysis of diagnostic biopsies predicts pathological response to neoadjuvant chemoradiotherapy of esophageal cancer.

OBJECTIVE: This study explored gene expression differences in predicting response to chemoradiotherapy in esophageal cancer. PURPOSE: A major pathological response to neoadjuvant chemoradiation is observed in about 40% of esophageal cancer patients and is associated with favorable outcomes. However, patients with tumors of similar histology, differentiation, and stage can have vastly different responses to the same neoadjuvant therapy. This dichotomy may be due to differences in the molecular genetic environment of the tumor cells. BACKGROUND DATA: Diagnostic biopsies were obtained from a training cohort of esophageal cancer patients (13), and extracted RNA was hybridized to genome expression microarrays. The resulting gene expression data was verified by qRT-PCR. In a larger, independent validation cohort (27), we examined differential gene expression by qRT-PCR. The ability of differentially-regulated genes to predict response to therapy was assessed in a multivariate leave-one-out cross-validation model. RESULTS: Although 411 genes were differentially expressed between normal and tumor tissue, only 103 genes were altered between responder and non-responder tumor; and 67 genes differentially expressed >2-fold. These included genes previously reported in esophageal cancer and a number of novel genes. In the validation cohort, 8 of 12 selected genes were significantly different between the response groups. In the predictive model, 5 of 8 genes could predict response to therapy with 95% accuracy in a subset (74%) of patients. CONCLUSIONS: This study has identified a gene microarray pattern and a set of genes associated with response to neoadjuvant chemoradiation in esophageal cancer. The potential of these genes as biomarkers of response to treatment warrants further investigation.

Hypoxia response element-driven cytosine deaminase/5-fluorocytosine gene therapy system: a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro.

BACKGROUND: We proposed to exploit hypoxia-inducible factor (HIF)-1alpha overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1alpha to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells. METHODS: We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evaluated in 22Rv1 and DU145 cells in vitro. RESULTS: The CD protein as selectively detected in lysates of transiently transfected 22Rv1 and DU145 cells following hypoxic exposure. This is the first evidence of GAPDH HREs being used to control a suicide gene therapy strategy. Detectable CD levels were sustained upon reoxygenation and prolonged hypoxic exposures. Hypoxia-induced chemoresistance to 5-FU was overcome in both cell lines treated with this suicide gene therapy approach. Hypoxic transfectants were sensitized to prodrug concentrations that were ten-fold lower than those that are clinically relevant. Moreover, the surviving fraction of reoxygenated transfectants could be further reduced with the concomitant delivery of clinically relevant single radiation doses. CONCLUSIONS: This strategy thus has the potential to sensitize the hypoxic compartment of prostate tumours and improve the outcome of current therapies.

Recognition of O6MeG lesions by MGMT and mismatch repair proficiency may be a prerequisite for low-dose radiation hypersensitivity.

Low-dose hyper-radiosensitivity (HRS) is the phenomenon whereby cells exposed to radiation doses of less than approximately 0.5 Gy exhibit increased cell killing relative to that predicted from back-extrapolating high-dose survival data using a linear-quadratic model. While the exact mechanism remains to be elucidated, the involvement of several molecular repair pathways has been documented. These processes in turn are also associated with the response of cells to O6-methylguanine (O6MeG) lesions. We propose a model in which the level of low-dose cell killing is determined by the efficiency of both pre-replicative repair by the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) and post-replicative repair by the DNA mismatch repair (MMR) system. We therefore hypothesized that the response of cells to low doses of radiation is dependent on the expression status of MGMT and MMR proteins. MMR (MSH2, MSH6, MLH1, PMS1, PMS2) and MGMT protein expression signatures were determined in a panel of normal (PWR1E, RWPE1) and malignant (22RV1, DU145, PC3) prostate cell lines and correlated with clonogenic survival and cell cycle analysis. PC3 and RWPE1 cells (HRS positive) were associated with MGMT and MMR proficiency, whereas HRS negative cell lines lacked expression of at least one (MGMT or MMR) protein. MGMT inactivation had no significant effect on cell survival. These results indicate a possible role for MMR-dependent processing of damage produced by low doses of radiation.

Increased spontaneous apoptosis, but not survivin expression, is associated with histomorphologic response to neoadjuvant chemoradiation in rectal cancer.

PURPOSE: Survivin has been shown to be an important mediator of cellular radioresistance in vitro. This study aims to compare survivin expression and apoptosis to histomorphologic responses to neoadjuvant radiochemotherapy (RCT) in rectal cancer. MATERIALS AND METHODS: Thirty-six pre-treatment biopsies were studied. Survivin mRNA and protein expression plus TUNEL staining for apoptosis was performed. Response to treatment was assessed using a 5-point tumour regression grade. RESULTS: Survivin expression was not found to be predictive of response to RCT (p = NS). In contrast, spontaneous apoptosis was significantly (p = 0.0051) associated with subsequent response to RCT. However, no association between survivin expression and levels of apoptosis could be identified. CONCLUSIONS: This in vivo study failed to support in vitro studies showing an association between survivin and response to chemotherapy and radiation therapy. These results caution against the translation of the in vitro properties of survivin into a clinical setting.

Breast cancer outcomes following a national initiative in Ireland to restructure delivery of services for symptomatic disease.

BACKGROUND: A national initiative in Ireland in 2000 defined 13 designated Units to provide care for symptomatic breast cancer, and resources, including an ability to develop audit programmes, were provided. In the absence of a national audit of breast cancer outcomes, the aim of this study is to provide a detailed report of one Unit's subsequent experience, in particular comparing process and outcome data with international norms and benchmarks, and to infer on the likely impact of the national initiative. METHODS: A 5-year prospective audit of patients presenting to the Symptomatic Breast Clinic from 2001 to 2005 was conducted. All cancer diagnoses were discussed at the Breast Multidisciplinary Conference, and all clinicopathological treatment details and follow-up information were entered by a full-time data manager. Overall survival was calculated using the Kaplan-Meier method. RESULTS: Eight hundred and thirty-nine patients were diagnosed through the clinic, 18 (2%) Stage 0, 169 (20%) Stage I, 380 (45%) Stage II, 142 (17%) Stage III, and 123 (15%) Stage IV. At a median follow-up of 35 months the overall 5-year survival was 71%, with 100%, 91%, 83%, 72%, and 11% survival for Stages 0-IV, respectively, and disease-specific survival of 82%. CONCLUSIONS: The process and outcome data are consistent with international benchmarks. These data from one designated centre support the national initiatives in Ireland to restructure breast services.

Predicting the response of localised oesophageal cancer to neo-adjuvant chemoradiation.

BACKGROUND: A complete pathological response to neo-adjuvant chemo-radiation for oesophageal cancer is associated with favourable survival. However, such a benefit is seen in the minority. If one could identify, at diagnosis, those patients who were unlikely to respond unnecessary toxicity could be avoided and alternative treatment can be considered. The aim of this review was to highlight predictive markers currently assessed and evaluate their clinical utility. METHODS: A systematic search of Pubmed and Google Scholar was performed using the following keywords; "neo-adjuvant", "oesophageal", "trimodality", "chemotherapy", "radiotherapy", "chemoradiation" and "predict". The original manuscripts were sourced for further articles of relevance. RESULTS: Conventional indices including tumour stage and grade seem unable to predict histological response. Immuno-histochemical markers have been extensively studied, but none has made its way into routine clinical practice. Global gene expression from fresh pre-treatment tissue using cDNA microarray has only recently been assessed, but shows considerable promise. Molecular imaging using FDG-PET seems to be able to predict response after neo-adjuvant chemoradiation has finished, but there is a paucity of data when such imaging is performed earlier. CONCLUSION: Currently there are no clinically useful predictors of response based on standard pathological assessment and immunohistochemistry. Genomics, proteomics and molecular imaging may hold promise.

COX-2 overexpression in pretreatment biopsies predicts response of rectal cancers to neoadjuvant radiochemotherapy.

PURPOSE: To determine the utility of COX-2 expression as a response predictor for patients with rectal cancer who are undergoing neoadjuvant radiochemotherapy (RCT). METHODS AND MATERIALS: Pretreatment biopsies (PTB) from 49 patients who underwent RCT were included. COX-2 and proliferation in PTB were assessed by immunohistochemistry (IHC) and apoptosis was detected by TUNEL stain. Response to treatment was assessed by a 5-point tumor-regression grade (TRG) based on the ratio of residual tumor to fibrosis. RESULTS: Good response (TRG 1+2), moderate response (TRG 3), and poor response (TRG 4+5) were seen in 21 patients (42%), 11 patients (22%), and 17 patients (34%), respectively. Patients with COX-2 overexpression in PTB were more likely to demonstrate moderate or poor response (TRG 3+4) to treatment than were those with normal COX-2 expression (p=0.026, chi-square test). Similarly, poor response was more likely if patients had low levels of spontaneous apoptosis in PTBs (p=0.0007, chi-square test). CONCLUSIONS: COX-2 overexpression and reduced apoptosis in PTB can predict poor response of rectal cancer to RCT. As COX-2 inhibitors are commercially available, their administration to patients who overexpress COX-2 warrants assessment in clinical trials in an attempt to increase overall response rates.

Prostate Cancer Unit Initiative in Europe: A position paper by the European School of Oncology.

The Prostate Cancer Programme of the European School of Oncology developed the concept of specialised interdisciplinary and multiprofessional prostate cancer care to be formalized in Prostate Cancer Units (PCU). After the publication in 2011 of the collaborative article "The Requirements of a Specialist Prostate Cancer Unit: A Discussion Paper from the European School of Oncology", in 2012 the PCU Initiative in Europe was launched. A multiprofessional Task Force of internationally recognized opinion leaders, among whom representatives of scientific societies, and patient advocates gathered to set standards for quality comprehensive prostate cancer care and designate care pathways in PCUs. The result was a consensus on 40 mandatory and recommended standards and items, covering several macro-areas, from general requirements to personnel to organization and case management. This position paper describes the relevant, feasible and applicable core criteria for defining PCUs in most European countries delivered by PCU Initiative in Europe Task Force.

Molecular pathology of prostate cancer: the key to identifying new biomarkers of disease.

Microarray technology has recently accelerated the study of the molecular events involved in prostate cancer, offering the prospect of more precise prognosis and new therapeutic strategies. This review summarises current knowledge of the molecular pathology of prostate cancer. The expression and function of numerous genes have been shown to be altered in prostate cancer. Many of these genes are involved in cell cycle regulation, steroid hormone metabolism or regulation of gene expression. The mechanisms by which androgen independence arises are discussed, including cross-activation, gene amplification and point mutations of the androgen receptor. Analysis of changes in the levels of expression of large numbers of genes during prostate cancer progression have provided a better understanding of the basis of the disease, yielding new molecular markers, such as hepsin, with potential use in diagnosis and prognosis.

Accelerated radiation therapy, seven fractions per week, for advanced head and neck cancer--a feasibility study.

AIM: The feasibility and improved efficacy of six conventional fractions per week has previously been proven in a Danish randomized trial. We tested the tolerance and efficacy of seven conventional fractions per week using a concomitant boost technique. METHODS: From September 1996 to May 1998, 20 patients with squamous cancer of the head and neck were treated with radiation alone. The site of disease was oropharynx in 35%, larynx in 30%, oral cavity 20%, and hypopharynx in 15%. All patients had stage III (10%)/IV (90%) disease. The planned total dose to gross disease was 66 Gy delivered in 33 fractions of 2 Gy each in 31 days. Large volumes were treated to 46 Gy, 2 Gy per fraction, once each morning, Monday-Friday. Boosts to gross disease consisted of 20 Gy in 10 fractions > or = 6 h after the morning dose on Tuesday and Thursday. RESULTS: Acute toxicity > or = grade 3 was mucous membrane 75%, pharynx 60%, skin 65%, and larynx 35%. One acute toxicity was fatal. Chronic toxicity > or = grade 3 (three patients) was mucous membrane 5%, pharynx 10%, skin 5%, salivary 15%, and larynx 5%. All patients with grade III or greater late toxicity had grade III acute toxicity in each toxicity category. At 30 months Kaplan-Meier survival is 55%, and local control is 39%. CONCLUSIONS: Without increasing resource utilization this scheme accelerates treatment by 30%. As expected acute toxicity is high but manageable. Chronic toxicity appears comparable to other altered fractionation strategies however the median follow up is only 30 months more toxicity may emerge as the data matures. We plan further trials using 1.8 Gy fractions to reduce toxicity.